Investigations of Mechanisms Mediating Contamination of Produce by Listeria, and Pre-Harvest Risk Factors for Campylobacter in Turkeys

<p>NON-TECHNICAL SUMMARY:<br/> Listeria monocytogenes remains a major contributor to severe illness and death due to foodborne infection in the United States. In 2011, the largest foodborne outbreak in the United States to date involved whole cantaloupe contaminated with Listeria, and several additional recent outbreaks have involved fresh produce. In spite of the clear public health and economic burden associated with Listeria-contaminated produce, our understanding of the underlying mechanisms is currently minimal. Further understanding is critically needed for development of novel intervention strategies to prevent colonization of produce with this pathogen. In this project we will follow microbiological and molecular approaches to elucidate these mechanisms for contamination of produce. The project will result in currently lacking baseline data and knowledge needed to
reduce Listeria-associated hazards of fresh produce. Campylobacter is a leading bacterial cause of human foodborne disease, with an estimated 0.8 million cases of illness each year in the United States alone. In addition to acute gastroenteritis, it is associated with potentially severe autoimmune complications. Poultry consumption is a major risk factor for human infections and farm-level control of Campylobacter colonization of poultry will be critical for reductions in human disease burden. However, pre-harvest control of Campylobacter will remain challenging and incomplete as long as we lack a good understanding of how Campylobacter is transmitted to poultry pre-harvest. It is critical that farm-level practices associated with high prevalence of Campylobacter become identified, and that effective training programs are made available to the poultry industry. The current project will
combine microbiological surveys, identification of potential pre-harvest risk practices, training and outreach in order to address these needs with a focus on the turkey industry. Turkeys have been understudied in terms of their colonization with Campylobacter, but may contribute importantly to Campylobacter-related food safety risks. Industry practices leading to turkeys free of Campylobacter, or with significantly reduced loads, would promote compliance with government-mandated Campylobacter standards for poultry and also result in a product with reduced potential to compromise food safety and human health.
<p>APPROACH:<br/> Objective 1: To assess relative produce colonization ability of a panel of strains of L. monocytogenes, including representatives of different lineages and sources. To determine possible associations between the ability of Listeria monocytogenes (LM) to contaminate produce and attributes such as lineage, serotype, and strain source, a carefully chosen panel of diverse will be employed. Three different produce types will be used: cantaloupe (rind and flesh), leafy greens (spinach and lettuce) and celery. The spot inoculation approach will be used for inoculations and inoculated produce will be kept at 5oC (28 days), 10oC (ten days) or 25oC (72 h). CFUs from three inoculated pieces at each time point will be determined on selective media (MOX). Data will be analyzed using SAS for analysis of variance and Duncan's multiple range tests to determine
significance of differences in regard to serotype, lineage and temperature. Objective 2: To identify and characterize genes of L. monocytogenes required for produce colonization (adherence, survival, growth). We will combine DNA sequencing and the screening of mariner-based mutant libraries to identify genes of LM required for (1) adherence and (2) survival and growth on produce. We will employ two different LM strains in these studies. Bioinformatics analysis of the sequence data (transposon junction sequences) will be performed to identify insertionally inactivated genes over-represented in non-adherent mutants relatively to the pool of mixed mutant libraries used as inoculum ("inoculum pool"). These genes will be candidates for adherence-required genes. Similar bioinformatics analysis will identify mutants in for genes essential for survival and growth on the produce. Choice of
genes for further analysis will be based on the statistical strength of the over-representation or under-representation of the insertions relative to their abundance in the inoculum pool. Objective 3: To assess the role of the genes on biofilm formation by L. monocytogenes on stainless steel and on produce. The deletion mutants and their wildtype parental counterparts will be analyzed in biofilms on stainless steel and on produce. Mutant and wild type strains will be grown at 25oC and used to inoculate washed, sterilized stainless steel coupons. Coupons will be incubated (5, 10 and 25oC) for variable lengths of time and bacteria will be enumerated. For biofilms on produce, LM will be inoculated onto cantaloupe rind and spinach leaves as described earlier. To determine the levels of LM in biofilms versus solitary cells, bacteria will be washed off the produce and filtered through a 5
µm polycarbonate filter. Solitary cells will be measured by plating the filtrate onto MOX, and biofilm-associated cells will be determined by sonication of the filter to remove and disperse aggregates, followed by plating onto MOX. Objective 4. Identify farm-level practices that contribute to either consistently low or consistently high prevalence of Campylobacter in turkey flocks pre-harvest We will enroll 10 Campylobacter-negative and 10 Campylobacter-positive farms, each with two successive flocks per year. Each flock will be sampled for Campylobacter (1) at the brooder stage (approx. 5 wks), (2) shortly after placement at the grow-out farm, (3) during the week immediately prior to processing, and (4) after evisceration at the processing plant. Farms will be identified upon consultation with company veterinarians who have precise data on date of flock placement and location of
the farms. Campylobacter in fecal droppings will be enumerated and characterized for species (C. jejuni or C. coli), antibiotic susceptibility profiles, and strain type (via PFGE and MLST). Upon our first visit to the farm we will perform a GAPS-like audit with special attention to practices that may place the farm at enhanced risk for contamination of the birds with Campylobacter. Statistical analysis using multivariable logistic regression will be done to identify relationships between high Campylobacter prevalence and specific risk practices identified by the GAPS-like audit of the farms. Similarly, practices with protective effect (associated with low prevalence or absence of Campylobacter) will be identified. Statistical analysis will also be done to assess the relationship between presence and numbers pre-harvest and at the processing plant post-evisceration. Suggestions based on
these analyses will be communicated to company veterinarians, farmers and other stakeholders as detailed below. Objective 5. Based on Campylobacter prevalence data and farm management assessments, develop training and outreach materials for the turkey industry. Training materials: Practices enhancing or reducing the risk for Campylobacter will be placed in an order of priority based on their odds ratio scores. These practices will be communicated to stakeholders (company veterinarians, flock technicians, workers and farmers) through training materials and relevant demonstrations. Training material will be placed at all extension offices in the state and will be also mailed to all turkey growers from the company veterinarians' office. In addition, they will be placed on the open-access web page constructed in association with the project. Visits to the individual farms will be done
by an Area Extension Agent, who has direct knowledge of the farms and the communities involved. During these visits specific risk practices will be pointed out and possible mitigations will be demonstrated. The Area Extension Agent will also schedule training sessions to groups of growers. Training sessions will include clarification of the risks involved in specific practices, and "how-to" discussions on implementation of the changes in the day-to-day-operation of the farm and the turkey house. Objective 6. Evaluate impact of research, training and outreach Evaluation of project effectiveness. During the early stages, the focus will be on monitoring and formative evaluation efforts. Program records will be used to assess whether key activities and interventions are implemented as intended (e.g., are targets enrolling in workshops). Furthermore, qualitative methods (e.g., interviews)
will be used to collect information from stakeholders/targets for the purpose of refining and improving the outreach and extension activities. Near the end of the project, the emphasis will shift to impact evaluation. More structured and quantitative methods (e.g., questionnaires) with appropriate controls will be used to assess effectiveness of the outreach and extension activities. Three main sets of data will be employed for the evaluation: (i) Evaluations (surveys) administered after each training session; (ii) GAPS-like farm audit data before and after administration of training materials and outreach efforts; and (iii) laboratory data pre-harvest and at the processing plant, pre- and post- the training materials and outreach. Additional tools will involve a mail survey and assessment of web page use. Research impact will be evaluated by metrics that include numbers of graduate and
undergraduate students who obtain research experience; numbers of undergraduate honors projects, theses or dissertations; numbers of presentations at scientific conferences; numbers of publications and citations in the literature.